Project Summary The long term goal of this project is to uncover the regulatory networks that control the Hippo signaling pathway in response to mechanical stimuli, which could lead to better treatments for cancer, and improved stem cell therapies. The canonical Hippo pathway consists of two tumor suppressor kinases called MST1/2 and LATS1/2. MST1/2 activate LATS1/2, which in turn phosphorylates and inhibits the transcriptional co-activator and oncogene YAP, causing it to relocalize from the nucleus to the cytoplasm. When in the nucleus, the YAP promotes cell survival and proliferation. YAP nuclear localization is regulated by diverse mechanical stimuli such as substrate stiffness, cell detachment, cell crowding, and stretch to control density dependent inhibition of growth, tissue repair and stem cell proliferation and differentiation. Although these mechanical stimuli appear to affect Hippo signaling through effects on the F-actin cytoskeleton, the molecular mechanism for how F-actin perturbations are translated into changes in Hippo pathway signaling is largely unknown. Here we will identify how mechanical forces act on the core Hippo pathway. In Specific Aim 1, we will determine how LATS1/2 activating kinases are regulated to turn on LATS1/2 in response to mechanical forces. In Specific Aim 2, we will test various models for how angiomotin proteins act as sensors to regulate multiple aspects of Hippo signaling in response to mechanical stimuli that affect F-actin levels. We will determine in Specific Aim 3 how TRIP6 regulates LATS1/2 activity in response to cellular tension across sheets of cells. Overall these studies will reveal key molecular pathways controling Hippo signaling in response to mechanical forces. These studies will have an important impact on our understanding of tumor suppression and tissue regeneration and may lead to ways to manipulate these important processes.